鸭疫里氏杆菌LC1和CX1株的分离鉴定及致病性分析

doi:10.11843/j.issn.0366-6964.2024.09.043

摘要/Abstract

摘要：

旨在对福建莆田某养殖场疑似感染鸭传染性浆膜炎患病番鸭病料进行菌株的分离、纯化、鉴定和致病性分析。采集10日龄患病番鸭的脑、肝组织进行菌株分离纯化、16S rDNA PCR鉴定和血清型鉴定, 并检测分离纯化菌株的致病性。结果得到2株11型鸭疫里氏杆菌, 分别将其命名为LC1和CX1。对2株分离株进行16S rDNA序列测序, 开展遗传进化分析和MLST分型, 发现LC1和CX1分离株16S rDNA遗传进化上与鸭疫里氏杆菌福建分离株RAf115和上海分离株Yb2处在同一进化分支上, 相似性为99.9%。测定其耐药性发现, 分离株对β-内酰胺类、氯霉素类及四环素敏感, 对大环内酯类和氨基糖苷类等抗菌素不敏感。进一步检测该分离株对雏番鸭致病性, 结果发现两株菌株(1.3×10⁶ CFU·mL^－1)的致死率为100%, 且病死鸭表现典型心包炎、肝周炎、气囊炎和脾肿大的鸭疫里氏杆菌病。病理结果发现, 致死鸭脾红白髓界线消失, 呈现大量坏死灶。此外, 两分离株致死鸭的心肌纤维呈撕裂状病变。综上, 本研究分离并鉴定2株11型鸭疫里氏杆菌LC1和CX1, MLST分型分别为ST111、ST114, 为鸭疫里氏杆菌病的防控及后续研究提供参考依据。

关键词: 鸭疫里氏杆菌, LC1, CX1, 遗传进化分析, 致病性

Abstract:

In currently study, LC1 and CX1 of Riemerella anatipestifer type 11 were isolated and purified from the brain and liver tissue of 10-day-old ducks, The aim of this study was to isolate, purify, identify and analyze the pathogenicity of the strains from the disease material of Muscovy ducks which suspected to be infected with infectious serositis from a farm in Putian, Fujian Province, China. Brain and liver tissues of 10-day-old diseased Muscovy ducks were collected for strain isolation and purification, and 16S rDNA PCR identification and serotyping were conducted, respectively. The isolates were subjected to 16S rDNA sequencing and genetic evolutionary analyses, and their drug resistance and pathogenicity to ducklings were determined. The 16S rDNA sequence sequencing and genetic evolution of these isolates were analyzed, and their drug resistance and pathogenicity to ducklings were further determined. (Results) The results showed that 2 strains of Riemerella anatipestifer type 11 were obtained and named as LC1 and CX1. The 16S rDNA sequences of LC1 and CX1 showed that the isolates were in the same evolutionary branch as the reference strains of Riemerella anatipestifer Fujian strain RAf115 and Shanghai strain Yb2, with 99.9% similarity. The isolates were sensitive to β-lactams, chloramphenicol, and tetracyclines, but less sensitive to macrolides and aminoglycosides. The pathogenicity assay indicated that the mortality of the two strains (1.3×10⁶ CFU·mL^-1) was 100%, and the lethal ducks showed the typical Riemer's disease with pericarditis, perihepatitis, and enlarged spleen.The pathology results of the HE staining showed that the red-white medullary boundary of the spleen of the lethal ducks disappeared, and a large number of necrotic foci were presented. In addition, myocardial fibers of lethal ducks from the two isolates showed tear-like lesions. These datas demonstrated that two strains of Riemerella anatipestifer type 11 LC1 and CX1 were isolated and characterized, MLST to be ST111 and ST114, respectively, which provide reference basis for the prevention and control of Riemerella anatipestifer disease and subsequent research.

Key words: Riemerella anatipestifer, LC1, CX1, genetic evolution analysis, pathogenicity

中图分类号:

S852.617

谢碧林, 林志敏, 林彬彬, 徐以娟, 林锋强, 闫露, 伍慧妮, 李翠婷, 周海欧, 李兆龙. 鸭疫里氏杆菌LC1和CX1株的分离鉴定及致病性分析[J]. 畜牧兽医学报, 2024, 55(9): 4196-4203.

Bilin XIE, Zhimin LIN, Binbin LIN, Yijuan XU, Fengqiang LIN, Lu YAN, Huini WU, Cuiting LI, Haiou ZHOU, Zhaolong LI. Isolation, Identification and Pathogenicity Analysis of Riemerella anatipestifer Strain LC1 and CX1[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4196-4203.

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名称Name	序列Sequence	长度/bp Length
RAdnaB-F	GAATGATTTCTTCCGAGACAGG	570
RAdnaB-R	AAAGCCTTACTTCGCCCGT
RAgroE-F	TCAAGAGACGCACTTAAAAGAGGTG	519
RAgroE-R	TGTACCTTTAGCCTCTTCCACAGTA
RAgyrA-F	GCCTCATAACTTATCGGAATC	797
RAgyrA-R	AATAGCTTTGTACTCATCACGG
RAmdh-F	ACAGCAGGGTCTCAAGTGG	532
RAmdh-R	TCTGGTCGCAAGCAATAGC
RAgluD-F	TTGTTTCCGCTCGCTCTAAG	769
RAgluD-R	TAATCCCGTTCATCGCCAC
RAgpi-F	GCGATAGATAGTATGTTTTCTGGAG	575
RAgpi-R	TCTACCACCAACCCAGTCC
RArpoB-F	TTAGATCCCATCAAGGCACG	686
RArpoB-R	GAGCAGTTGGCAGGTCAGTT

抗菌素Antibiotic	判定标准/mm Criterion			分离株Isolates
抗菌素Antibiotic	R	I	S	LC1	CX1
青霉素G Penicillin G	≤17	18~20	≥21	24	25
氨苄西林Ampicillin	≤13	14~16	≥17	17	20
阿莫西林Amoxicillin	≤13	14~17	≥18	30	32
头孢曲松Ceftriaxone	≤14	15~17	≥18	31	33
头孢吡肟Cefepime	≤14	15~17	≥18	33	31
庆大霉素Gentamicin	≤12	13~14	≥15	0	0
阿米卡星Amikacin	≤14	15~16	≥17	0	0
新霉素Neomycin	≤12	13~16	≥17	0	0
四环素Tetracycline	≤14	15~18	≥19	30	35
强力霉素Doxycycline	≤12	13~15	≥16	31	32
红霉素Erythromycin	≤13	14~22	≥23	0	0
恩诺沙星Enrofloxacin	≤15	16~20	≥21	0	13
环丙沙星Ciprofloxacin	≤15	16~20	≥21	0	15
复方新诺明Compound sulfamethoxazole	≤9	10~16	≥17	0	0
多黏菌素B Polymyxin B	≤7	8~11	≥12	0	0
氟苯尼考Florfenicol	≤12	13~17	≥18	34	30

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